Infection by human T-cell lymphotropic virus type I (HTLV-I) is characterized by a long clinical latency; only a minor proportion (1 to 4%) of infected individuals develop disease such as adult T-cell leukemia (ATL) or HTLV-I-associated myelopathy (HAM), also known as tropical spastic paraparesis (3,30,36). ATL is an aggressive malignancy of mature T-cells frequently associated with infilterative skin lesions, lymphadenopathy, and hypercalcemia. In contrast, HAM is a chronic slowly progressive neurological condition characterized by motor weakness along with bladder and bowl dysfunction. The factors responsible for progression from latency to productive infection and manifestation of disease are still undefined. It has been suggested that activation of the lymphoid cells in the host plays a crucial role in triggering viral expression (31). Indeed, antigenic stimulation of HTLV-I infected cells in vitro results in activation of viral gene expression and replication (6,25). Collectively, differences in both functional and clinical characteristics of ATL and HAM suggest that altered viral strains, clonal expansion of infected cells or host immune responsiveness may be responsible for these clinical outcomes.
While the genetic predisposition to the disease has not been completely ruled out, genotypic analysis of the viral strains from individuals with ATL, HAM or asymptomatic carriers have demonstrated minimal sequence variations, suggesting that the viral strain itself does not appear to play a major role in the pathogenesis. Furthermore, clonal proliferation of HTLV-I-infected T-cells has not been found to be a unique feature of HAM, since asymptomatic HTLV-I-infected carriers also had frequent clonal expansion of HTLV-I-infected cells. Analysis of humoral immune responses has demonstrated elevated levels of HTLV-I-specific antibodies, however, no preferential recognition of immunodominant epitopes of the gag and env proteins in any clinical group suggested that immune responsiveness to HTLV-I structural proteins may not be associated with the disease manifestation. Alternatively, specific cellular immune responses might be responsible for the development of HTLV-I-specific disease. Indeed, elevated levels of both in vitro spontaneous proliferation (12,13) and HTLV-1-specific cytotoxic T-lymphocytes (CTL) of peripheral blood mononuclear cells (PBMC) from patients with HAM have been reported (14-16) and it has been proposed that CTL against HTLV-I proteins may contribute to the disease pathogenesis. However, recent studies demonstrating the presence of activated CTL in asymptomatic carriers 917) raises some uncertainty about their significance in the pathogenesis of the disease.
Previous studies (12,37) report a correlation between antibody to p40.sup.tax protein of human T cell leukemia virus 1 and infectivity, the prevalence of antibody to p42 of HTLV-I among ATL patients in comparison with healthy carriers, and hyperimmune responsiveness to HTLV-I antigens in HAM patients (19,23, respectively). It is also reported that almost all of HAM and approx 50% of ATL and asymptomatic donors have been shown to contain anti-tax antibodies (48).
Further, HTLV-I proviral DNA and RNA are detected more frequently in patients with HAM than in those of asymptomatic carriers. Recent studies have indicated that patients with HAM not only have increased HTLV-I proviral DNA load (7,14), but also enhanced viral transcription (8,33). More recently, it has been suggested that concomitant expression of mRNA specific for the regulatory proteins, pX and the upregulation of interleukin-2 (IL-2) and the .alpha. subunit of IL-2 receptor (IL-2R.alpha.) in patients with HAM, is essential for maintaining a state of lymphocyte activation in HAM, thereby contributing to the pathogenesis of this disease (33). The data on pX expression among ATL are inconsistent; one group found increased expression (13) but others did not detect any message (22,33).
The pX gene region of HTLV-I encodes for at least two regulatory proteins, termed tax (p40.sup.x) and rex (p27.sup.x-III) (30). Both of these proteins are translated in different open reading frames from the double spliced mRNA, both are positive regulators of gene expression, and both have been implicated in viral leukemogenesis. The tax protein is a transcriptional activator that not only augments activity of its own long terminal repeat (LTR), but also induces the expression of several cellular genes, in particular those involved in T-cell activation and proliferation (30,33). In contrast, the rex protein acts at a posttranscriptional level, leading to expression of the structural gene products necessary for virion assembly (30).
The amino-terminal region of the tax protein of HTLV-I has previously been shown to contain functional domains required for the trans-activation of its own long terminal repeat (LTR) through a cyclic-AMP response element pathway, as well as selected cellular and heterologous viral promoters through activation of NF-kB (30). Mutational analysis of tax has further demonstrated that the amino acids .sup.123 ThrLeu contained within the tax8 epitope are crucial for trans-activation through both NF-kB and CRE pathways (29) and may play a role in disease pathogenesis. However, whether the carboxyl-terminus of tax protein is implicated in the transactivation function remains unclear (27,29).
Regarding the rex protein, basic amino acids at the amino terminus have been shown to be responsible both for the nuclear-nucleolar localization and for the protein's function mediated through the cis-acting RNA sequences (rex response element [RxRE]).2 Thus, while the functional domains of tax and rex have been identified (27,29,34), not much is known about the immunogenic regions of these proteins, in particular during natural infection.
Anti-tax antibodies have been shown to be associated with the increased incidence of vertical and sexual transmission of the HTLV-I (4,12). The antigenicity of the carboxyl terminal domain of tax protein has previously been suggested by virtue of rabbit antisera to the carboxyl terminus peptide or mouse monoclonal antibodies reacting with the native tax protein (29,32). A recombinant peptide of tax (Rp-F.sup.329-353) has recently been shown to react with 42.8% of the serum specimens (20). Most of the studies analyzing anti-tax responses have utilized recombinant tax protein expressed in various expression vectors in a WB format (4,5,12,14,21,28,37,42-44). However, the results of such preparations can vary depending upon the concentration and purity of the recombinant protein used.
Thus, there exists a need for a synthetic peptide based assays to provide a method of diagnosing HAM and ATL.